Anthocyanins are naturally occuring pigments belonging to the group of flavonoids, a subclass of the polyphenol family. They are common components of the human diet, as they are present in many foods, fruits and vegetables, especially in berries and red wine. There were more studies conducted on effect of processing and storage on changes and stability of colors of anthocyanins in foods such as fruits and also for their use as natural colorants. Besides, the interest on anthocyanins is still growing also owing to their strong antioxidant activity against many chronic diseases, numerous studies about their medicinal, therapeutical and nutritional value were also conducted. There are pieces of evidence regarding the positive association of their intake with healthy biological effects. They act as antioxidants both in the foodstuffs in which they are found and in the organism that take in foods rich in anthocyanins. Many efforts have been carried out to develop new analytical techniques for identification and quantification of anthocyanins in plant materials, as well as their effects in vivo and in vitro. With this in mind, an overview to general considerations concerning (i) polyphenol and flavonoid history; (ii) chemical structure, color and intake of anthocyanins and (iii) sample preparation and extraction methods are presented in this chapter.
- sample preparation
The progress in the last years in the interdisciplinary fields of chemistry, separation science, biology and pharmacy has boosted the natural product chemistry research [1–4], providing a valuable information about many classes of naturally occurring dietary phytochemicals. Among these phytochemicals, polyphenols are the worldwide redox-active secondary metabolites of a phenolic nature [5–12]. The importance of secondary metabolites and their crucial role in many important functional aspects of plant life was recognized for the first time in the second-half of the ninetieth century by Julius Sachs (1873) [13, 14]. Polyphenols are natural compounds occurring in plants [15–18], including foods such as fruits, vegetables, cereals, tea, coffee and wine.
The study mainly focuses on organoleptical properties of polyphenols  and their physiological importance to plants . Later, polyphenols are found to be recognized by their nutritional value, since they may help reduce the risk of chronic diseases [1, 21–24] and, in general, have a positive effect on health, because of their free radical scavenging capacity [25–27], which, among other biological effects, increases antioxidant activity and prevents cellular oxidation.
The research on phenolic compounds is mainly focused on anthocyanins [28–29], natural pigments and common components of the human diet (foods, fruits and vegetables, especially in berries and in red wine), As they provide for much of the red to blue pigmentation of flowers and fruits and have physiological functions in vegetative tissues. Their biosynthetic pathway has been the subject of much research and the associated biosynthetic and regulatory genes are well defined. Besides considerable interest in coloring properties of anthocyanins, they have also attracted attention due to their antioxidant activity [30–34] and their property is closely related, to a large extent, with their chemical structure. The pH-dependent ground-state chemistry of anthocyanins is extremely rich. In the past 20 years, the health benefits of anthocyanins have become the subject of intensive research.
This chapter intends to reflect the interdisciplinary nature of the research that is currently carried out in anthocyanin pigments through an update of the state-of-art of a series of previously published reviews on this field in the year 2012 [28, 29, 38, 39]. First, general considerations concerning polyphenols with emphasis on their role as secondary metabolites are made. Flavonoid classification, structure, biological activities, databases, intake and dietary sources are also contemplated. Second, aspects of anthocyanin concerning its early history and chemical structure, color and intake are dealt. It should be noted that anthocyanins are readily distinguished from other flavonoids as they undergo rearrangements in response to pH. The antioxidant activity of anthocyanins is depending on their chemical structure. Finally, special attention is paid to analytical methodologies involved in the isolation, determination and characterization of bioactive polyphenols in plants, fruits and vegetables, herbal drugs, medicinal plants and wines, including sample-handling strategies, a feature of analysis often ignored. The use of nonthermal technologies in the assisted extraction of anthocyanins will be covered in future reports.
Plant phenols are among the most abundant and widely represented class of existing plant natural products  thanks to the continuous evolution of new genes brought about by gene duplication and mutation and subsequent recruitment and adaptation to specific functions.
The amino acids phenylalanine and tyrosine (derived from the shikimic acid pathway) are the most common origin of polyphenols [41, 42]. Chemically, polyphenols belong to four main classes (Figure 1): flavonoids, phenolic acids (hydroxy derivatives of benzoic acid and cinnamic acid, i.e., p-hydroxybenzoic, protocatechuic, vanillic and syringic acids) and their esters (chlorogenic, caftaric, coutaric and fertaric acids), stilbenes (resveratrol, pterostilbene, piceatannol), characterized by a double bond (1,2-diarylethene) connecting the phenolic rings and lignans (pinoresinol, podophyllotoxin, steganacin), characterized by a 1,4-diarylbutane structure, i.e., having 2-phenylpropane units. Flavonoids and phenolic acids account for 60 and 30%, respectively, of the total dietary polyphenols .
When the phenolic molecules are not attached to sugar moieties are known as the aglycone form; while those molecules conjugated with one or more sugar residues are called glycosides. Most phenolic compounds are found in nature associated with mono- or polysaccharides or functional derivatives  such as esters or methyl esters, varying widely in their hydroxylation pattern and can be glycosylated or acylated.
Despite the fact that most of the literature on phenolic compounds focuses mainly on those found in fruits, vegetables, wines and teas. However, many phenolic compounds present in fruits and vegetables (phenolic acids and flavonoids) are also found in cereals [45, 46].
For decades, this family of compounds has attracted the attention  since three British scientists open the door to understand separation, structural elucidation and taxonomical distribution of phenolic compounds. As mentioned above, traditionally, the interest was focused on organoleptical properties of polyphenol such as color, astringency, bitterness, astringency and a range of other tactile or “mouth feel” characteristics [48, 49], as well as their physiological role in plants in the reproduction, pathogenesis and symbiosis. In last decades, polyphenols are increasingly recognized due their nutritional value, since they may help reduce the risk of chronic diseases [50–53]. The capacity of phenolic compounds to trap free radicals depends upon their structure, in particular of the hydrogen atoms of the aromatic group that can be transferred to the free radicals [54, 55] and of the capacity of the aromatic compound to cope with the uncoupling of electrons as a result of the surrounding displacement of the electron-π system . The polyphenols are still gaining attention.
Although the percentage of absorbed natural polyphenols is usually quite low , researchers have seen a large quantity of metabolites of polyphenols in the form of simple phenolic acids in the blood. The amount and form in which plant phenolic substances are administered influence greatly the physiological effects connected with their consumption . About 1 g of polyphenols per day is commonly ingested with foods, being the most abundant antioxidant in the diet (about 10 times higher than the intake of vitamin C and 100 times that of vitamin E) [28, 29, 58]. The daily intake of polyphenols is difficult to estimate and depend on several factors. In the literature there are about 1000 peer-reviewed publications  concerning the polyphenol content in food. Recently, the construction and application of a database with polyphenols content in foods has facilitated this task . The Institute Nationale de la Recherche Agronomique  has developed a new Phenol-Explorer database  covering over 60,000 foods useful to epidemiologists, food scientists and food manufacturers. The content of polyphenols of the 100 richest dietary sources can range from 15,000 mg per 100 g in cloves to 10 mg per 100 mL in rose wine . EuroFir  is another database to build national food composition in different countries within the framework of the European Food Information Resources Network (i.e., Spanish  and Irish  databases).
The flavonoids constitute the largest group of polyphenols (of low molecular weight) and are considered to be responsible for the color and taste of many fruits and vegetables [65, 66]. Since first identified in the mid-1800s, more than 9000 flavonoid structures have been described, with formulas, references and biological information [67, 68] and the list is constantly increase. These include over 600 different naturally occurring anthocyanins  that are widely distributed among at least 27 families, 73 genera and innumerable species.
Many of these compounds are yellow in color, as the Latin root suggests. They are present particularly in the epidermis of leaves and the skin of fruits [9, 70]. Flavonoids can accumulate in vacuolar compartments, or be secreted, for example, as part of root exudates. Most attracting is the accumulation of flavonoids on the surface of leaves and flowers . Flavonoids play roles in many facets of plant physiology  and their influence on the transport of the plant hormone auxin is one of their most important roles.
Around 5000 of the flavonoids studied have antioxidant activity. Because the number of phytochemicals already identified is only a small part of those that exist in nature, there is considerable interest in new methods [37, 72–78] of separation, isolation and characterization of polyphenol structures from foods.
Concerning the chemical structures of flavonoids in which two aromatic rings are present that linked by three carbons in an oxygenated heterocycle, i.e., a flavan (2-phenyl-benzo-γ pyran) nucleus consisting of two benzene rings combined with an oxygen-containing pyran ring, the parent compound bearing a tricyclic (C6-C3-C6) skeleton. The heterocyclic benzopyran ring is known as the C ring, the fused aromatic ring as the A ring and the phenyl constituent as the B ring. The structural differences in each flavonoid family result from variations in the number/substitution pattern of the hydroxyl and methoxy groups [28, 79, 80], as well as different glycosylation patterns and the presence of a C2-C3 double bond in the heterocycle pyran ring.
Compounds are classified according to differences in their heterocycle (C ring): flavonols, flavones (catechins), flavanones, chalcones, dihydrochalcones and dihidroflavonols, anthocyanins and isoflavonoids (isoflavones) [29, 55], varying in the oxidation state (degree of saturation) of the heterocyclic central pyran ring. When unsaturation is present, the geometry of the molecule is planar, as in the case of anthocyanins, flavones and flavonols.
Flavonoids usually occur as glycosides in plants, reflecting a biological strategy increasing their polarity and necessary for storage in the plant cell vacuoles and decreasing their reactivity to interact with macromolecules [81–83]. While flavan-3-ols (catechins and theaflavins) are present in either a free form or as gallic acid esters (e.g., in tea). The glycosidic linkages appear to be important for the absorption of flavonoids .
Williams and Grayer  have stated: “Flavonoids continue to capture the interest of scientists from many different disciplines because of their structural diversity, biological and ecological significance (e.g. the colored pigments in many flower petals) and health promoting and anti-cancer properties.” Recent advances in genomics, proteomics and metabolomics provide new approaches in the field of flavonoids in plant: protection against oxidative diseases, ability to modulate the activity of various enzymes and interactions with specific receptors are among the most significant health benefits [67, 85].
Flavonoids of dietary significance are present in edible plants in widely varying combinations [86, 87]. Unlike traditional vitamins, flavonoids are not essential for short-term well-being. The daily intake is almost at the same level as the sum of other antioxidants, including carotene, vitamin C and vitamin E, can range from several hundred mg up to 1–2 g  and although flavonoids are not essential for short-term may exhibit potential health benefits at modest long-term intake [85, 89, 90]. Table 1 [91–98] shows the dietary sources of flavonoids. Great differences in flavonoid intake and food sources were observed between a large Mediterranean cohort and non-Mediterranean populations (U.S. and Finland as non-Mediterranean countries) . The mean intake for a Spanish population was 313 mg/day . Estimated per capita daily flavonoid intake is 182 and 177 mg for the UK and Ireland, respectively .
|Flavonoid subclass||Prominent food flavonoids||Typical rich food sources|
|Bilberries, black and red currants, blueberries, cherries, chokeberries, elderberries, grapes, strawberries, pomegranate|
|Apples, pears, strawberries, tomatoes, cinnamon|
|Apples, blueberries, grapes, onions, lettuces, red wine, tea, chocolate, apricots, sour cherries, grape juice, mint|
|Flavanonols||Taxifolin or dihydroquercetin|
Aromaderin or dihydrokaempherol
|Grapes, red onion, açai palm|
|Citrus fruits and juices, peppermint|
|Apples, bean, blueberries, buck wheat, cranberries, endive, leeks, broccoli, lettuces, onions, olive, pepper, tomatoes|
|Citrus fruits, celery, parsley, spinachs, rutin, olives, artichoke|
|Soybeans, grape seed/skin, chick peas, black beans, green peas|
|Xanthones||Mangostin||Mango, mangosteen, bark of pear, apples, cherries|
Anthocyanins are the largest group of phenolic pigments and the most important group of water-soluble pigments in plants [68, 69, 101–104], responsible for the red, purple and blue colors found in many fruits, vegetables, cereal grains and flowers, being odorless and nearly flavorless and contributing to taste as a moderately astringent sensation. Anthocyanins are almost universally found in higher plants (occurring in about 30 families), but in general anthocyanins seem to be absent  in the liverworts, algae and other lower plants, although some of them have been identified in mosses and ferns. Figure 2 shows a picture of plant species rich in anthocyanins [105–107].
Anthocyanins are found mainly in the skin, except for certain types of red fruit , in which they also occur in the flesh (cherries and strawberries).
Anthocyanin biosynthesis was one of the first branches of the general propanoid metabolism [41, 108, 109], for which biosynthetic enzymes and corresponding transcription factors were identified, given the ease of visualization and control of mutants and genetic imbalances.
Anthocyanins have characteristic physicochemical properties that confer them its unique color and stability [30, 32, 110–112]. They are highly reactive molecules and thus sensitive to degradation reactions. Oxygen, temperature, light, enzymes and pH are among the factors that may affect anthocyanins chemistry and, consequently, their stability and color . In the following, aspects of anthocyanins concerning its chemical structure, color, antioxidant activity and intake are dealt.
4.1. Chemical structure
Chemically, anthocyanins are glycosylated polyhydroxy or polymethoxy derivatives of 2-phenylbenzopyrilium [68, 103, 114], usually with molecular weights ranging from 400 to 1200 (medium-size biomolecules) and containing two benzyl rings (A and B). Anthocyanins usually contain a single glucoside unit, but many anthocyanins contain two, three, or more sugars attached at multiple positions , or occurring as oligosaccharide side chains. Intensity and type of the color of anthocyanins is affected by the number of hydroxyl and methoxyl groups [68, 115]: if more hydroxyl groups are present then the color goes toward a more bluish shade; and redness is increased if more methoxyl groups are present. The major anthocyanins are shown [116, 117] in Figure 3 and Table 2.
|Anthocyanidin||Abbrev.||R1||R2||λmax (nm)*||Some of the produced colors|
|R3 = H||R3 = gluc|
|Delphinidin||Dp||OH||OH||546||541||Purple, mauve and blue|
|Cyanidin||Cy||OH||H||535||530||Magenta and crimson|
Anthocyanins mainly exist in glycosidic forms in fruits and with the exception of blueberries, fruits usually contain anthocyanin derived from only one or two of the aglycone bases. Grapes offer a richer anthocyanin profile than many other fruits  (red grapes may contain a mixture of more than 20 pigments [85, 119]. Various berries and black currants are the anthocyanin-richest fruits [120–122]. The eggplant is only one common vegetable  that contains a high level of anthocyanins.
The anthocyanins are all amphoteric [32, 34] forming salts with either acids or bases. In addition, anthocyanins occur in plants as salts (indicated by the positive charges on the heterocyclic ring) and their color in plant cells depends mainly upon their mode of combination. The conjugated bonds in their structures (light-conjugated double bonds carrying a positive charge), which absorb light at about 500 nm, are the basis of the bright red, blue and purple color of fruits and vegetables  as well as the autumn foliage of deciduous trees. Every color except green has been observed (either natural or synthetic), depending on aspects such as a kind of substituent present in the B-ring, the local pH, the state of aggregation of the anthocyanins, complexation by organic molecules, or, as in the case of blue color , complexation by metal cations.
In spite of the increasingly large number of structures, they are derived from only about 30 different anthocyanidins [69, 126], most of them are based on cyanidin (31%), delphinidin (22%), or pelargonidin (18%). The other common anthocyanidins (peonidin, malvidin and petunidin), which contain methoxy group(s) on their B-ring (Figure 4), represent together 21% of the isolated anthocyanins. One new methylated anthocyanidin, 7-O-methylcyanidin, five new desoxyanthocyanidins and a novel type of anthocyanidin called pyroanthocyanidin [11, 127] have also been reported. In spite of the structural diversity of anthocyanins, the three nonmethylated anthocyanidins are the most widespread in nature , which are present in 80% of pigmented leaves, 69% of fruits and 50% of flowers.
4.2. Antioxidant activity
The relationship between diet and health has been known since ancient times and recent studies demonstrated the relevance of many food components in modulating health . Due to anthocyanin’s positive charge (Figure 3), the number and arrangement of aromatic hydroxyl groups, the extent of structural conjugation and the presence of electron-donating and electron-withdrawing substituents in the ring structure made anthocyanins very effective donors of hydrogen to highly reactive free radicals (such as superoxide (O2−), singlet oxygen (1O2), peroxide (RCOO⋅), hydrogen peroxide (H2O2) and hydroxyl radical (OH⋅) and reactive nitrogen species in a terminator reaction) and, thereby preventing further radical formation [55, 91, 129]. This effect protect cells from oxidative damage, which leads to aging and various diseases [102, 130–133], such as cancer, neurological and cardiovascular diseases, inflammation, diabetes and bacterial infections. The antioxidant capacity of phenolic compounds is also attributed to chelate metal ions involving in the production of free radicals , thereby reducing metal-induced peroxidation. Anthocyanin bioavailability has been the subject [135–138] of recent reviews.
Laboratory-based evidence was provided (the potential health benefits of anthocyanins . Consumption of diets rich in natural bioactive components (i.e., fruits and vegetables) as an alternative to pharmaceutical medication has been a subject [104, 140, 141] of considerable interest to researchers. In recent investigations carried out in population-based anthocyanins have been linked to a decrease in the incidence of several diseases, such as diabetes mellitus, cancer and cardiovascular diseases.
Anthocyanins are the pigment compounds responsible for pale yellow, orange, red, magenta, violet and blue colors . Carotenoids and betalains confer yellow and red colors , although only the families of Caryophyllales (except for Caryophyllaceae and Molluginaceae) produce betalains. Up to now, no plants producing both anthocyanins and betalains  have been discovered. Anthocyanins, carotenoids and other pigments contribute to the UV patterns that are visible to insects and serve  to signal flowers that are attractive to pollinators. Figure 5 shows a schematic representation of the biochemical process involving anthocyanins in color plants .
In food chemistry, anthocyanins have been studied [68, 147, 148] in relation to changes and stability of colors in foods such as fruits during processing and storage and also for their use as natural colorants. Indeed, many types of anthocyanin food colorants have been developed and are now available to customize the appearance of foods. In horticulture, color conversion of flower pigments has become possible by new findings of anthocyanin research. Creation of flowers in new colors enriches our life; for example, the creation of blue roses  is a noteworthy achievement. Genetic engineering is the key technology for converting flower color and it became possible after the discovery of genes involved in anthocyanin biosynthesis and elucidation of their expression mechanisms.
Additionally, color may act as a “fingerprint” of a food product, being related to its flavor and at the same time  an estimate of its overall quality. In this sense, special attention is paid to the application of anthocyanin analysis in classification of wine . Anthocyanins can be used as markers to classify wines according to the grape variety [49, 151], although this requires a complex separation with very high chromatographic efficiency, together with advanced statistical methods, especially when dealing with aged red wines, because of the formation  of pyranoanthocyanins (formed through the reaction of anthocyanins with small molecules).
Copigmentation is almost always a variation toward blueness. This phenomenon induced by the presence of substances [153, 154] that are themselves colorless or only slightly colored in wine has received considerable attention [155, 156]. The basic role of copigments is to protect  the colored flavylium cation from the nucleophilic attack of the water molecule. The copigmentation complexes are easily disrupted by dilution returning to the pH-dependent equilibria among the structural forms of anthocyanins. It has been suggested that in acylated anthocyanins, the acyl groups interact with the basic anthocyanin structure, thus avoiding the formation of the hydrated species. This is the basis of characterizing  the color due to copigmentation. Copigmentation of flavonoids other than anthocyanins is also possible , but it is either a rare or an understudied phenomenon.
Anthocyanins are widely ingested by humans, mainly due to consumption  of red fruits (like berries and red grapes), vegetables such as red cabbage, red wines, cereals and purple corn. Accurate estimation of anthocyanin contents in foods and daily intake is critical in food science, nutrition and other related research fields. The type and concentration of anthocyanins differ widely among different fruits and vegetables. Intake levels of anthocyanins varies widely with region, season and among individuals with different social, cultural and educational backgrounds. High intake levels of anthocyanins can be achieved with the regular consumption of fruits (blueberries, blackberries, raspberries, strawberries, red grapes and saskatoon berries). Depending on nutritional habits, the daily intake has been estimated in the range  from several milligrams to hundreds of milligrams per person, while the consumption of other phytonutrients such as carotenoids, vitamin E and vitamin C are estimated at 5, 12 and 90 mg/day, respectively.
Regular consumers of red wine are likely to have  significantly higher intakes. A glass of red wine provides around 115 mg of polyphenols, contributing toward a total intake  of phenolic compounds of 1171 mg/person/day. In the United States, an average daily intake of anthocyanins has been estimated  at 215 mg during the summer and 180 mg during the winter. Wu et al.  estimated that the mean daily intake of anthocyanins is 12.5 mg/person in the United States; such a huge difference of the total anthocyanin daily intake estimation must result from different food intake data. The influence of methodological differences in the assessment, as well as nutritional, social and cultural differences of the investigated populations, may also explain the wide range of anthocyanin consumption estimated by different authors. Anthocyanin intake in the German young shows differences between girls and boys , decreasing from young childhood to adolescence.
Up to now, anthocyanins have not been detected in processed food such as canned food, bread, or cereals. Also, although prepared baby food containing blueberries, rich in anthocyanins, are expected to find these compounds, analyses have hardly detected them . In young infants  the anthocyanidin intake was found to be zero.
Due to antioxidant and other potential beneficial properties, grapes, various berries, red cabbage and other anthocyanin-rich foods are becoming more popular. Berry extracts are also being commercialized as nutraceuticals and dietary supplements  to meet consumer demands.
Currently, there is no recommended intake level of anthocyanins for optimal health or to avoid adverse effects; however, future research and continued consumer interest will undoubtedly present opportunities for pursing dietary guidance recommendations.
5. Sample preparation and extraction of anthocyanins
The presence of phytochemical bioactive compounds in food and dietary supplements poses difficult problems in connection with the optimization of their extraction process and determination. Aspects related to Ref.  the complexity of sample matrices, the presence of varying forms of bioactive substances and interaction with other components need to be solved. A number of factors including pH, metal ions, complex formation, light, temperature, enzymes, sugars, oxygen and ascorbic acid exert influence  on the stability of anthocyanins. The role of analytical chemistry is vital in this context, e.g., [34, 76, 91, 167–171] promoting advances in separation science.
Most phenolic compounds are made up of only C, H and O, differing in some cases even by only one atom and in many others by constitutional or stereochemical isomers. The identification of species proves thus be difficult because of subtle structural changes, being necessary for this purpose to apply  complementary techniques. Anthocyanins may be forming part of complexes, may be present in matrices of a complex nature and may appear in distinct equilibrium forms [30, 32, 34], depending of the pH of the medium. Acid dissociation, rate constant and tautomerization constants are of great importance in the analysis of bioactive compounds and in the interpretation of their mechanisms of action.
Time-consuming processes are involved  in the isolation, purification and determination of the structures of anthocyanins, which must be accomplished with care. However, there is no universal sample pretreatment technique applicable to all kind of samples. The primary steps required, sampling, sample preservation and sample preparation, are not always properly documented  in the analytical literature.
5.1. Sample preparation
A number of strategies are used [37, 174, 175] for the characterization of phenolic samples in plant materials. In any case extraction techniques and semipreparative isolation methods are usually applied prior to the separation and quantification steps. After the first operations of drying and of powdering the plant material, it follows : (i) a previous extraction step of the plant materials as well as a preliminary consequent purification step; (ii) fractionation of the mixture in order to isolate pure pigments and (iii) the final characterization and identification of pure anthocyanins compounds (Table 3).
|Sample pretreatment||Air-drying, freeze drying|
|Solid extraction (SE)|
|Liquid-liquid extraction (LL)|
|Microwave-assisted extraction (MAE)|
|Ultrasound-assisted extraction (UAE)|
|Pressurized fluid extraction (PFE)|
|- pressurized liquid extraction (PLE/ASE)|
|- subcritical water extraction (SWE)|
|- supercritical fluid extraction (SPE)|
|Enzyme-assisted extraction (EAE)|
|Solid phase microextraction (SPME)|
|Membrane extraction (ME)|
|High hydrostatic pressure (HHP)|
|Electric fields (EF)|
|Purification||Solid phase extraction (SPE)|
|Column chromatography (CC)|
|Countercurrent chromatography (CCC)|
|Gas chromatography (GC)|
|Liquid chromatography techniques (LC)|
|Mass spectrometry (MS)|
|Nuclear magnetic resonance (NMR)|
|Capillary electrophoresis (CE)|
|Thin layer chromatography (TLC)|
|GC-MS, LC-MS, LC-DAD-ES-MS/MS,|
|CE-MS, LC-NMR, others|
In order to avoid sample oxidation, thermal degradation, chemical and biochemical changes under mild extraction conditions  are recommended and drying, lyophilized, or frozen samples should be used. The deterioration processes of the compounds may be avoided by the addition of antioxidants compounds, by using inert atmospheres or working in the absence of light. However, no definitive procedure for storage and collection has been established.
To determine either the target analytes (in their various conjugated forms) or the aglycones is an important question [175, 178] to answer. When dealing with plant, food products and biological matrices, the instant conjugates are usually search, whereas in the other instances it is necessary to carry out a preliminary hydrolysis, e.g., an enzymatic or chemical (acidic or alkaline) treatment. Intentional hydrolysis for obtaining the aglycones of some flavonoid or derivatization of some fatty acids to esters is sometimes intentionally incorporated  to the extraction process.
Extraction represents an important phase [34, 38, 39, 79, 169] in the isolation, identification and utilization of anthocyanins. Anthocyanins are usually recovered by mean of a solvent extraction procedure. Parameters such as solvent-extraction kind and its composition, liquid-to-solid ratio, extraction time and temperature require  proper optimization. The flavylium cation form of anthocyanins is red and stable in a highly acidic medium. Thus, the extraction solution should be enough (slightly) acid to maintain the flavylium cation form , but not so much as to cause partial hydrolysis of the acyl moieties in acylated anthocyanins. Protocols of extraction and analysis of plant materials and biological fluids are, however, difficult to accomplish because of  the structural diversity of anthocyanins and their susceptibility to heat, pH, metal complexes and copigmentation.
In the same way as flavonoids, in general, anthocyanins possess aromatic rings that contains polar substituent groups (─OH, ─C═O, or ─OCH3) and glucosyl residues, which altogether [28, 29] constitute a polar molecule. Flavonoid glycosides are of a more polar nature, whereas aglicones are extracted either with alcohols or alcohol-water mixtures. Cold acidified solvents (polar organic solvents, water) under mild conditions [167, 182] are used for the extraction of anthocyanins. The organic solvent usually used is methanol. However, solvents such as acetone, ethanol, or acetonitrile may be used. These solvents system denature the cell membranes  also dissolving and stabilizing the anthocyanins. Acetic acid at about 7% or trifluoroacetic acid at about 3% are usually used; the organic solvent content  varying from 50 to 100% in the mixture. When a mineral acid is used it may assist  to the loss of the attached acyl group. Sulfurous water also allows  the reduction of organic solvent and cost extraction.
Phytochemical recovery of a good antioxidant from various sources may be achieved by using solvent extraction. The conventional solvent extraction procedure suffers from the drawback of requiring subsequent extraction and cleanup prior analysis. In addition, health and safety risks are associated with the use of large amounts of organic solvents, being on the other hand environmentally unfriendly. A modern trend toward : (1) the use of samples smaller in size, volume, or organic solvent content; (2) an extraction with increasing selectivity or specificity; (3) improved recoveries and reproducibility; (4) greater automation facilities. A variety of modern techniques have been developed for this purpose, including solid phase extraction (SPE), countercurrent chromatography (CCC), microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), pressurized hot water extraction (PHWE) and high hydrostatic pressure extraction (HHP), among others. Selected applications of sample preparation techniques on anthocyanin compounds are listed in Table 4 [185–213], an extension of applications previously published by the authors in . The applications of other novel nonthermal techniques will be the subject of further study. Figure 6 shows a schematic representation of a highly separation and purification methodology based on a macroporus polymeric adsorbent for the determination of anthocyanins in bilberry .
|Solid phase extraction|
|Type of matrix/analyte||Extraction/cleanup technique||Sorbent||Final analysis||References|
|Petals of the oboshibana plant (||Sonication with 10% TFA aqueous solution/SPE||Discovery DPA-6S and DSC-SCX||UPLC-DAD|||
|Urine, serum and feces/anthocyanin metabolites||Addition of a preservative (10% w/v ascorbate in 0.5 mM EDTA) prior to SPE||Strata-X SPE cartridges||LC-MS/MS|||
|Red wines/anthocyanins and derived pigments||SPE||Zip-Tip® pipette tips filled with C18 stationary phase||ToF-MS|||
|Chinese black rice wine/phenolic constituents||Acidification at pH 2 with HCl/SPE||Oasis HLB||LC-MS/MS|||
|Wine lees/anthocyanidins, proanthocyanidins and anthocyanins||Liquid phase: SPE with filtration previous. Solid residue: MAE with a mixture 60:40 (v/v) ethanol-water||HySpere C8 EC cartridges (end-capped silica-based octyl phase, particle size 10 μm, 10 × 2 mm i.d.)||LC-MS/MS|||
|Hybrid grape/monomeric, nonanthocyanins, condensed tannins and anthocyanins||Acidification with HCl 0.01 M and centrifugation/SPE||Oasis HLB||LC-DAD|||
|Countercurrent chromatographic methods|
|Type of matrix/analyte||Solvent system||Elution mode/comment||References|
|Crude mulberry/cyanidin-3-glucoside and cyanidin-3-rutinoside||MTBE/n-butanol/acetonitrile/0.01% TFA (1:3:1:5, v/v)||The upper phase was used as the stationary phase and the lower phase as the mobile phase|||
|Blue honeysuckle fruits/cyanidin 3-glucoside||MTBE/n-butanol/acetonitrile/water/TFA (2:2:1:5:0.01, v/v). HSCCC||Head–tail elution mode with the upper organic phase as stationary phase|||
|Mulberry fruit/anthocyanins||MTBE, 1-butanol, acetonitrile, water and TFA (10:30:10:50:0.05; %, v/v)||Stationary phase: upper organic phase. Mobile phase: lower aqueous phase. The elution was in “head to tail” mode|||
|Petals of ||n-butanol/MTBE/acetonitrile/0.1% aqueous TFA (0.715:1.0:0.134:1.592, v/v/v/v). HSCCC||The lower phase in the solvent separator was pumped into the mobile phase bottle, the pumps for pumping MTBE, n-butanol, acetonitrile and 0.1% TFA aqueous were set to a certain flow rate, the solvents were mixed and separated into two layers; and after rinsing the solvent separator, the upper phase flowed into the stationary phase bottle|||
|Adsorbents and eluting agents|
|Solution (target compounds)||Adsorbent||Eluting and/or regenerating agent||References|
|Purple-fleshed potato/anthocyanins||Amberlite XAD-7HP||75 vol.% acidic (7 vol.% acetic acid) aqueous ethanol|||
|Blackberries/anthocyanins||Polyamide resin||Deionized water and ethanol (0.1% v/v HCl, pH = 3)|||
|Bilberry based/anthocyanins||Copolymerization of divinylbenzene and ethylene glycol dimethyl acrylate||Ethanol|||
|Blueberries/anthocyanins and polyphenols||FPX66 resin||3 bed volumes of 95% ethanol|||
|Jamun (||Amberlite XAD7HP||Aqueous acidified ethanol (above 40%, v/v)|||
|Aronia melanocarpa berries/antioxidant phenolics||XAD 7HP resin||Ethanol-water mixtures|||
|Strawberry/aroma compounds and Anthocyanins||Cross-linked acrylonitrile-co-divinylbenzene (AN/DVB)||Methanol|||
|Muscadine (||FPX-66 resin||Three bed volumes of aqueous ethanol (70%)|||
|Black carrot/anthocyanins||T-10-coded||1% acetic acid solution and methanol|||
|Pressurized fluid extraction|
|Matrix/compounds||Solvent||T (°C)||Pressure/cycles||Extraction time||Technique||References|
|Blackberry (||Ethanol:water 50% v/v||100||7.5 Mpa||55 min||UPLC-QToF-MS and UPLC-UV-Vis|||
|Red grape pomace/anthocyanins||Ethanol:water 50% v/v||120||90 bar||90 min||LC-Uv-vis|||
|Solanum stenotomun peel/antioxidants||Ethanol in water acidified to pH 2.6||80||100 bar||3 h||LC-Uv-vis|||
|Jabuticaba skins/anthocyanins||Ethanol||553 K||5 Mpa||31 min||Uv-vis|||
|Purple-fleshed sweet potato genotypes/anthocyanins||Acetic acid:methanol:water mixture of 7:75:18% (v/v)||100||1500 psi||20 min||LC-Uv-vis|||
|Microwave assisted extraction|
|Blackberry/anthocyanins||Ethanol was used as solvent. Microwave power of 469 W, a solvent concentration of 52%, a liquid–solid ratio of 25 g mL−1 and a time of 4 min||MAE was found to be the most effective method for improving the yield and antioxidant capacity among the other methods tested||Vis|||
|Blueberry leaves/anthocyanins||80 mL of solvent consisting of 30% ethanol and 1.5 M citric acid combination in a ratio of 97:3 (v/v)||Time extraction and microwave power level were observed to be significant factors affecting the extraction of phenolic compounds. MAE was the best extraction method among MAE, ultrasonic extraction and 24-h room temperature extraction||Folin-Ciocalteu reagent (spectrophotometric method)|||
|Black currant marc/anthocyanins||Maximum yields of anthocyanins were achieved at pH2 with an extraction time of 10 min with a microwave power of 700 W.||A significant reduction of extraction time was achieved using MAE and the final anthocyanin concentration in the solvent phase of MAE increased by 20 % compared to the conventional extraction||LC-DAD|||
|Mulberry/anthocyanins||59.6% acidified methanol, 425 W power, 25 (v/w) liquid-to-solid ratio and 132 s time||In comparison with conventional extraction, MAE is more rapid and efficient for extracting anthocyanins from mulberry||LC-MS|||
6. Identification and quantification: a primer
The identification of anthocyanins has a critical role in taxonomic  and adulteration  studies, besides anthocyanins might replay synthetic days. HPLC, especially in the reversed phase, is the most widely used separation technique. Due to pH-dependent interconversions among various molecular forms of the anthocyanins, a highly acidic mobile phase (pH < 2) is required to ensure that they are maintained predominantly in the flavylium cationic form for maximum chromatographic efficiency. However, even at low pH [31, 32, 35, 110] some interconversion between the anthocyanin flavylium cationic and carbinol pseudobasic forms occurs.
Regarding chromatographic detection techniques for the study of anthocyanins we have [168, 180, 216, 217] diode array detection (DAD) and MS or tandem mass spectrometry (MS/MS) among the most widely used. Spectroscopy is the main technique used due to its simplicity and low cost providing very useful qualitative and quantitative information (anthocyanins have a specific and intense absorbance band in the range of 520–560 nm) [218–220], however the difficulty in obtaining reference compounds and the spectral similarities of the anthocyanins represent important drawbacks.
Various MS instruments, as well as the advances in nuclear magnetic resonance (NMR) have given a fresh impetus to anthocyanin analysis [78, 83, 221]. MS/MS is particularly suited for structure elucidation and compound identification [217, 222, 223] since information pertaining to the aglycone moiety, type and number of sugars and other substituents can be obtained and many of the previously proposed reaction mechanisms for the formation of polymeric anthocyanins and other new pigments have been verified. NMR identification of anthocyanin compounds  offers new promising approaches for analysis of complex phenolic mixtures. NMR is based primarily on the analysis of 1H NMR spectra but important structural information can also be provided by 13C NMR  and, especially for compounds that have many quaternary carbons, by combining homo and heteronuclear 2D and 3D techniques. However, the relatively high capital costs are still an impediment  to their routine use in enforcement laboratories, a fact that must be taken into consideration.
The almost universal distribution of anthocyanins in flowering plants makes them also suitable for chemotaxonomic considerations  both at the family and genus level. Differential anthocyanins profiles may be used  for the detection and adulteration in specific commodities of berry fruit products. In the last few years, special attention has been paid  to the isolation and characterization of compounds that may delay the onset of aging, as occurs with some berry phenolics. The extremely low levels of anthocyanins usually present in biological samples [57, 168, 181, 186] (blood plasma and body tissues) possess further challenges to their identification and quantification, together with the lack of commercially available anthocyanin standards.
7. Final comments
In last decades, polyphenol chemistry has experienced an explosion of knowledge, being anthocyanins one of the most widely studied groups [27, 226, 227], due to its great potential for practical applications in various fields, contributing in addition this to obtain a better understanding of the chemistry of life.
Anthocyanins occur in all plant tissues including leaves, stems, roots, flowers and fruits imparting color. Anthocyanins are responsible for the red, purple and dark blue colors of many fruits and berries [68, 228–230]. Anthocyanins have antioxidant activity [55, 129, 231–233] preventing radical formation. These nontoxic natural pigments have received considerable attention from such as food, pharmaceutical and nutritional industries due to their potential applications in color-processed food and medicines [31, 33, 147, 234] which may replace synthetic dyes.
It was only a few decades ago that anthocyanins were regarded as highly degradable compounds and the research studies mainly were focused on their chemical structures, color stability, use as food constituents and changes in foods during storage. Anthocyanins are now recognized as food constituents with potential health benefits [102, 131] and research related to these properties has markedly progressed at the molecular level. Anthocyanins will continue to attract researchers across various disciplines, including those involved in the creation of new flower varieties with novel colors. Research on the health benefits of anthocyanins will provide information  on underlying molecular mechanisms and absorption and metabolism. Moreover, once these benefits are proven in humans, development of foods and dietary supplements in a capsule form [164, 235, 236] can be accelerated to promote the proven functions, i.e. berry extracts are being commercialized as nutraceuticals and as dietary supplements to fulfill consumer demands.
The development of analytical techniques to determine the identity and quantities of anthocyanins in natural products, as well as their effects
Solvent extraction involving the use of acidic solvents has been the most commonly used method [36, 79] for the recovery of diverse compounds found in flavonoids, including anthocyanins. The traditional solid-liquid or liquid-liquid extraction offers good recovery. Nevertheless, they are often described as laborious, time and solvent consuming and prone to errors. However, in recent years there are trends toward other environmentally and economically friendlier extraction techniques [38, 39, 110, 167, 184, 206, 238] using a smaller amount of (nontoxic) solvents and sample sizes, reducing working time and increasing selectivity, specificity, recovery and potential of automation. MAE, SFE, PLE, or PHWE are among the greener techniques that have experienced a large increase in recent years to extract anthocyanins from plant material and other samples.
Diode array detection
High hydrostatic pressure extraction
High-performance liquid chromatography
High-speed countercurrent chromatography
Triple quadrupole mass spectrometer
Methyl tert-butyl ether
Nuclear magnetic resonance
Pressurized hot water extraction
Pressurized liquid extraction
Quadrupole-time of flight
Supercritical fluid extraction
Solid phase extraction
Ultra performance liquid chromatography
Zhang H, Tsao R. Dietary polyphenols, oxidative stress and antioxidant and anti-inflammatory effects. Curr. Opin. Food Sci. 2016; 8:33–42.
Bohn T, McDougall GJ, Alegría A, Alminger M, Arrigoni E, Aura A-M, Brito C, Cilla A, El SN, Karakaya S, Martínez-Cuesta MC, Santos CN. Mind the gap-deficits in our knowledge of aspects impacting the bioavailability of phytochemicals and their metabolites. A position paper focusing on carotenoids and polyphenols. Mol. Nutr. Food Res. 2015; 59:1307–1323.
Ziyatdinova GK, Budnikov HC. Natural phenolic antioxidants in bioanalytical chemistry: state of the art and prospects of development. Russ. Chem. Rev. 2015; 84(2):194–224.
Li RH. Health-promoting components of fruits and vegetables in the diet. Adv. Nutr. 2013; 4(3):384S–392S.
Ferrazzano GF, Amato I, Ingenito A, Zarrelli A, Pinto G, Pollio A. Plant polyphenols and their anti-carcinogenic properties: a review. Molecules. 2011; 16(2):1486–1507.
Burton-Freeman B. Postprandial metabolic events and fruit derived phenolics: a review. Br. J. Nutr. 2010; 104(S3):S1–S14.
Chong MF-F, Macdonald R, Lovergrove JA. Fruit polyphenols and CVD risk: a review of human intervention studies. Br. J. Nutr. 2010; 104(S3):S28–S39.
Crozier A, del Rio D, Clifford MN. Bioavailability of dietary flavonoids and phenolic compounds. Mol. Aspects Med. 2010; 31(6):446–467.
Crozier A, Jaganath IB, Clifford MN. Dietary phenolics: chemistry, bioavailability and effects on health. Nat. Prod. Rep. 2009; 26(8):1001–1043.
Martin KR, Appel CL. Polyphenols as dietary supplements. A double-edged sword. Nutr. Diet. Suppl. 2010; 2:1–12.
Pereira DM, Valentao P, Pereira JA, Andrade PB. Phenolics: from chemistry to biology. Molecules. 2009; 14(6):2202–2211.
Whiting DA. Natural phenolic compounds 1900-2000: a bird’s eye view of a century’s chemistry. Nat. Prod. Rep. 2001; 18(6):583–606.
Neumann KH, Kumar A, Imani J. Plant cell and tissue culture – a tool in biotechnology: basic and application. In Principles and Practice. Chapter 10, Berlin: Springer-Verlag, 2009. pp. 181–182.
Hartmann T. From waste products to ecochemicals: fifty years research of plant secondary metabolism. Phytochem. 2007; 68(22–24):2831–2846.
Shahidi F, Ambigaipalan P. Phenolic and polyphenolics in foods, beverages and spices: antioxidant activity and health effects – a review. J. Funct. Foods 2015; 18:820–827.
Lima GPP, Vianello F, Correa CR, da Silva-Campos RA, Borguini, MG. Polyphenols in fruits and vegetables and its effect on human health. Food Nutr. Sci. 2014; 5:1065–1082.
Cicerale S, Lucas L, Keast R. Biological activities of phenolic compounds present in virgin olive oil. Int. J. Molec. Sci. 2010; 11(2):458–479.
González-Gallego J, García-Mediavilla V, Sánchez-Campos S, Tuñon MJ. Fruit polyphenols, immunity and inflammation. Br. J. Nutr. 2010; 104(S3):S15–S27.
Cheynier V. Polyphenols in foods are more complex than often thought. Am. J. Clin. Nutr. 2005; 81:223S–229S.
Grotewold E. The genetics and biochemistry of floral pigments. Annu. Rev. Plant Biol. 2006; 57:761–780.
Joseph SV, Edirisinghe I, Burton-Freeman B. Fruit polyphenols: a review of anti-inflammatory effects in humans. Crit. Rev. Food Sci. Nutr. 2016; 56(3):419–444.
Khurana S, Venkataraman K, Hollingsworth A, Piche M, Tai TC. Polyphenols: benefits to the cardiovascular system in health and in aging. Nutrients. 2013; 5:3779–3827.
Peluso I, Miglio C, Morabito G, Ioannone F, Serafini M. Flavonoids and immune function in human: a systematic review. Crit. Rev. Food Sci. Nutr. 2015; 55(3):383–395.
Umeno A, Horie M, Murotomi K, Nakajima Y, Yoshida Y. Antioxidative and antidiabetic effects of natural polyphenols and isoflavones. Molecules. 2016; 21:708–723.
Halake K, Birajdar M, Lee J. Structural implications of polyphenolic antioxidants. J. Ind. Eng. Chem. 2016; 35:1–7.
Martin SN, Barros L, Ferreira ICR. In vivo antioxidant activity of phenolic compounds: facts and gaps. Trends Food Sci. Technol. 2016; 28:1–12.
Quideau S, Deffieux D, Douat-Casassus C, Pouységu L. Plant polyphenols: chemical properties, biological activities and synthesis. Angew. Chem. Int. Ed. 2011; 50(3):586–621.
Martín Bueno J, Ramos-Escudero F, Sáez-Plaza P, Muñoz AM, Navas MJ, Asuero AG. Analysis and antioxidant capacity of anthocyanin pigments. Part I: general considerations concerning polyphenols and flavonoids. Crit. Rev. Anal. Chem. 2012; 42:102–125.
Martín Bueno J, Sáez-Plaza P, Ramos-Escudero F, Jímenez AM, Fett R, Asuero AG. Analysis and antioxidant capacity of anthocyanin pigments. Part II: chemical structure, color and intake of anthocyanins. Crit. Rev. Anal. Chem. 2012; 42:126–151.
Pina F, Oliveira J, de Freitas V. Anthocyanins and derivatives are more than flavylium cations. Tetrahedron. 2015; 71:3107–3114.
Pina F. Chemical applications of anthocyanins and related compounds. A source of bioinspiration. J. Agric. Food Chem. 2014; 62(39):6885–6897.
Pina F, Melo MJ, Laia CAT, Parola J, Lima JC. Chemistry and applications of flavylium compounds: a hanfull of colours. Chem. Soc. Rev. 2012; 41:896–908.
Santos-Buelga C, Mateus N, de Freytas V. Anthocyanins, plant pigments and beyond. J. Agric. Food Chem. 2014; 62:6879–6884.
Silva VO, Freitas AA, Maçanita AL, Quina FH. Chemistry and photochemistry of natural plant pigments: the anthocyanins. J. Phys. Org. Chem. 2016;2 9:594–599. DOI: 10.1002/poc.3534.
Alberts P, Stander MA, de Villiers A. Advanced ultra high pressure liquid chromatography-tandem mass spectrometric method for the screening of red wine anthocyanins and derived pigments. J. Chromatogr. A. 2012; 1235:92–102.
Giusti MM, Jing P. Analysis of anthocyanins, In Food Colorants: Chemical and Functional Properties, Socaciu, C. (Ed.), Chapter 6. Boca Raton, FL: Taylor and Francis, 2008. pp. 479–506.
Gleichenghagen M, Schieber A. Current challenges in polyphenol analytical chemistry. Curr. Opin. Food Sci. 2016; 7:43–49.
Navas MJ, Jiménez-Moreno AM, Martín Bueno J, Sáez-Plaza P, Asuero AG. Analysis and antioxidant capacity of anthocyanin pigments. Part III: an introduction to sample preparation and extraction. Crit. Rev. Anal. Chem. 2012; 42:284–312.
Navas MJ, Jiménez-Moreno AM, Martín Bueno J, Sáez-Plaza P, Asuero AG. Analysis and antioxidant capacity of anthocyanin pigments. Part IV: extraction of anthocyanins. Crit. Rev. Anal. Chem. 2012; 42:313–342.
Boudet AM. Evolution and current status of research in phenolic compounds. Phytochem. 2007; 68(22–24):2722–2735.
Vogt T. Phenylpropanoid biosynthesis. Mol. Plant. 2010; 3(1):2–20.
Pourcel L, Routaboul JM, Cheynier V, Lepiniec L, Debeaujon I. Flavonoid oxidation in plants: from biochemical properties to physiological functions. Trends Plant Sci. 2007; 12(1):29–36.
Nichenametla SN, Taruscio TG, Barney DL, Exon JH. A review of the effects and mechanisms of polyphenolic in cancer. Crit. Rev. Food Sci. Nutr. 2006; 46(2):161–183.
Kren V. Glycoside vs aglycon: the role of glycosidic residue in biological activity. In Glycoscience. Fraser-Reid B, Tasuka K, Thiem J. (Eds.), Berlin: Springer-Verlag, 2008.
Masisi K, Beta T, Moghadasian MH. Antioxidant properties of diverse cereal grains: a review on in vitro and in vivo studies. Food Chem. 2016; 196:90–97.
Dykes L, Rooney LW. Phenolic compounds in cereal grains and their health benefits. Cereal Foods World. 2007; 52(3):105–111.
Robins RJ, Gibbons S, Marston A. The fiftieth anniversary meeting of the phytochemical society of Europe: Churchill College Cambridge. 11–14 April 2007: Highlights in the Evolution of Phytochemistry. Phytochem. 2007; 68(22–24):2699–2704.
Mateus N. The chemistry of wine flavors – polyphenols. (in Portuguese) Revista Real Academia Gallega de Ciencias. 2009; 28:5–22.
Zimman A, Waterhouse AL, Kennedy JA. Short history of red wine color. ACS Symposium Series No. 1886, Chapter 1. Washington, DC: ACS, 2004. pp. 1–6.
Valdes L, Cuervo A, Salazar N, Ruas-Madiedo P, Gueimonde M, Gonzalez S. The relationship between phenolic compounds from diet and microbiota: impact on human health. Food Funct. 2015; 6:2424–2439.
Ciriminna R, Meneguzzo F, Fidalgo A, Ilharco LM, Pagliaro M. Extraction, benefits and valorization of olive polyphenols. Eur. J. Lipid Sci. Technol. 2016; 118:503–511.
Rigacci S, Stefani M. Nutraceutical properties of olive oil polyphenols. An itinerary from cultured cells through animal models to humans. Int. J. Mol. Sci. 2016; 17:843–871.
Sarkar D, Shelty K. Metabolic stimulation of plant phenolics for food preservation and health. Ann. Rev. Food Sci. Technol. 2014; 5:395–413.
Croft KD. Dietary polyphenols: antioxidants or not?. Arch. Biochem. Biophys. 2016; 595:120–124.
Leopoldini M, Russo N, Toscano M. The molecular basis of working mechanism of natural polyphenol antioxidants. Food Chem. 2011; 125(2):288–306.
Forester SC, Waterhouse AL. Metabolites are key to understanding health effects of wine polyphenolics. J. Nutr. 2009; 139(9):1824S–1832S.
Vacek J, Ulrichova J, Klejdus B, Simanek V. Analytical methods and strategies in the study of plant polyphenols in clinical samples. Anal. Methods. 2010; 2(6):604–613.
Scalbert A, Manach C, Morand C, Rémésy C, Jiménez L. Dietary polyphenols and the prevention of diseases. Crit. Rev. Food Sci. Nutr. 2005; 45(4):287–306.
Neveu V, Perez-Jimenez J, Vos F, Crespy V, du Chaffaut L, Du Mennen L, Knox C, Eisner R, Cruz J, Wishart D, Scalbert A. Phenol-Explorer: an online comprehensive data base on polyphenol contents in foods. Database: J. Biol. Databases Curat (Oxford). 2010; doi:10.1093/database/bap024.
Scalbert A. INRA, Phenol-Explorer: Database on Polyphenol Content in Foods; http://www.phenol-explorer.eu
Perez Jimenez J, Neveu V, Vos F, Scalbert A. Identification of the 100 richest dietary sources of polyphenols: an application of the Phenol-Explorer Database. Eur. J. Clin. Nutr. 2010; 64(Suppl. 3):S112–S120.
Finglas PM, Denny AR. EuroFIR approach to develop tools to assure the quality of the data compilation process. Food Chem. 2009; 113(3):759–767
Burgos MAM, Martinez-Victoria I, Milá R, Farrán A, Farre R, Ros G, Yago MD, Audi N, Santana C, Millán MBL, López SR, Mañas M, Martinez-Victoria E. Building a unified Spanish food database according to EuroFIR specifications. Food Chem. 2009; 113(3):784–788.
Black CJ, Ireland J, Moller A, Roe M, Walton J, Flynn A, Finglas PM, Kiely M. Development of an on-line Irish food composition database for nutrients. J. Food Compos. Anal. 2011; 24(7):1017–1023.
Hoensch HP, Oertel R. The value of flavonoids for the human nutrition: short review and perspectives. Clin. Nutr. Exp. 2015; 3:8–14.
Grotewold, E. (Ed.) The Science of Flavonoids. New York: Springer, 2006.
Fraga CG. (Ed.) Plant Phenolics and Human Health: Biochemistry, Nutrition and Pharmacology. New York: Wiley, 2010.
He J, Giusti M. Anthocyanins: natural colorants with health-promoting properties. Ann. Rev. Food Sci. Technol. 2010; 1(1):163–187.
Andersen OM, Jordheim M. The anthocyanins. In Flavonoids: Chemistry, Biochemistry and Applications. Andersen OM, Markham KR. (Eds.), Boca Raton, FL: Taylor and Francis, 2006. pp. 471–551.
Onylagha JC, Grotewold E. The biology and structural distribution of surface flavonoids. Recent Res. Develop. Plant Sci. 2004; 2:53–71.
Buer CS, Imin N, Djordjevic A. Flavonoids: new roles for old molecules. J. Integr. Plant Biol. 2010; 52(1):96–111.
Casassa LF, Haberlson JF. Extraction, evolution and sensory impact of phenolic compounds during red wine maceration. Ann. Rev. Food Sci. Technol. 2014; 5:83–109.
de Villiers A, Venter P, Pasch H. Recent advances and trends in the liquid-chromatography-mass spectrometry analysis of flavonoids. J. Chromatogr. A. 2016; 1430:16–78.
Khoddami A, Wilkes, MA, Roberts TH. Techniques for analysis of plant phenolic compounds. Molecules. 2013; 18:2328–2375.
Lorrain B, Ky I, Pechamat L, Teissedre P-L. Evolution of analysis of polyphenols from grapes, wines and extracts. Molecules. 2013; 18:1076–1100.
Motilva M-J, Serra A, Macià A. Analysis of food polyphenols by ultra high-performance liquid chromatography coupled to mass spectrometry: an overview. J. Chromatogr. A. 2013; 1292:66–82.
Szultka M, Papaj K, Rusin A, Szeja W, Buszewski B. Determination of flavonoids and their metabolites by chromatographic techniques. Trends Anal. Chem. 2013; 47:47–67.
Ziaullah HP, Rupasinghe V. Application of NMR spectroscopy in plant polyphenols associated with human health. Chapter 1. In Applications of NMR Spectroscopy. Bentham Science Publishers. Ltd. Published by Elsevier Inc. Sharjah, United Arab Emirates.Vol. 2, 2015. pp. 3–92.
Andersen OM, Markham KR. Flavonoids, Chemistry, Biochemistry and Applications. Boca Raton, FL: Taylor and Francis, 2006.
Kumar S, Pandey AK. Chemistry and biological activities of flavonoids: an overview. Sci. World J. 2013; 2013. http://dx.doi.org/10.1155/2013/162750.
Xiao J, Capanoglu E, Jassbi AR, Miron A. Advance on the flavonoid C-glycosides and health benefits. Crit. Rev. Food Sci. Nutr. 2016; 56:S29–S45.
Agati G, Azzarello E, Pollastri S, Tattini M. Flavonoids as antioxidants in plants: location and functional significance. Plant Sci. 2012; 196:67–76.
Corradini E, Foglia, P, Giansanti, P, Gubbiotti, R, Samperi, R, Lagana, A. Flavonoids: chemical properties and analytical methodologies of identification and quantitation in foods and plants. Nat. Prod. Res. 2011; 25(5):469–495.
Williams CA, Grayer RJ. Anthocyanins and other flavonoids. Nat. Prod. Rep. 2004; 21(4):539–573.
Jaganatah IB, Crozier A. Dietary Flavonoids and phenolic compounds, In Plant Phenolics and Human Health: Biochemistry, Nutrition and Pharmacology. Fraga CG. (Ed.) New York: Wiley, 2010. pp. 1–50.
Bhagwat S, Haytowitz DB, Wasswa-Kintu SI, Holden JM. USDA develop is a database for flavonoids to assess dietary intakes. Procedia Food Sci. 2013; 2:81–96.
Biesaga M, Pyrzynska K. Analytical procedures for determination of quercetin and its glycosides in plant material. Crit. Rev. Anal. Chem. 2009; 39(2)95–107.
Pan M-H, Lai C-S, Ho C-T. Anti-inflammatory activity of natural dietary flavonoids. Food Funct. 2010; 1(1):15–31.
Ahmad A, Kaleem M, Ahmed Z, Shafia H. Therapeutic potential of flavonoids and their mechanism of action against microbial and viral infections. A review. Food Res. Int. 2015; 77:221–235.
Chen L, Teng HT, Xie Z, Cao H, Cheang WS, Skalicka-Woniak K, Georgiev MI, Xiao J. Modifications of dietary flavonoids towards improved bioactivity: an update on structure-activity relationship. Crit. Rev. Food Sci. Technol. 2016; dx.doi.org/10.1080/10408398.2016.1196334
Ignat I, Volf I, Popa VI. A critical review of methods for characterisation of polyphenolic compounds in fruits and vegetables. Food Chem. 2011; 126(4):1821–1835.
Han X, Shen T, Lou H. Dietary polyphenols and their biological significance. Int. J. Mol. Sci. 2007; 8(9):950–988.
Naczk M, Shahidi F. Phenolics in cereals, fruits and vegetables: occurrence, extraction and analysis. J. Pharm. Biomed. Anal. 2006; 41(5):1523–1542.
Manach C, Donovan JL. Pharmacokinetics and metabolism of dietary flavonoids in humans. Free Radical Res. 2004; 38(8):771–785.
Beecher GR. Overview of dietary flavonoids: nomenclature, occurrence and intake. J. Nutr. 2003; 133(10):3248S–3254S.
Aherne SA, O’Brien NM. Dietary flavonols: chemistry, food content and metabolism. Nutrition. 2002; 18(1):75–81.
Duthie GG, Duthie SJ, Kyle JAM. Plant polyphenols in cancer and heart disease: implications as nutritional antioxidants. Nutr. Res. Rev. 2000; 13(1):79–106.
Rice-Evans C, Spencer JP, Schroeter H, Rechner AR. Bioavailability of flavonoids and potential bioactive forms in vivo. Drug Metabol. Drug Interact. 2000; 17(1–4):291–310.
Zamora-Ros R, Andres-Lacueva C, Lamuela-Reventos RM, Berenguer T, Jakszyn P, Barricate A, Ardanaz E, Amiano R, Dorronsoro M, Larrañaga N, Martinez C, Sanchez MJ, Navarro C, Chiriaque MD, Tormo MJ, Quiros JR, Gonzalez CA. Estimation of dietary sources and flavonoids intake in a Spanish adult population (EPIC-Spain). J. Am. Dietetic Assoc. 2010; 110(3):390–398.
Beking K, Vieira A. An assessment of dietary flavonoids intake in the UK and Ireland. Int. J. Food Sci. Nutr. 2011; 62(1):17–19.
Fang J. Classification of fruits based on anthocyanin types and relevance to their health effects. Nutrition. 2015; 31:1301–1306.
Wallace TC, Giusti MM. Anthocyanins in Health and Disease. New York, NY: CRC Press, 2014.
Gould K, Davies K, Winefields C. (Eds.) Anthocyanins, Biosynthesis, Functions and Applications. New York: Springer-Verlag, 2009.
Motohashi N, Sakagami H. Functionability of anthocyanins as alternative medicine. Topics Heterocyclic Chem. 2008; 15:1–48.
Borghesi E, González-Miret ML, Escudero-Gilete ML, Malorgio F, Heredia FJ, Meléndez-Martínez AJ. Effects of salinity stress on carotenoids, anthocyanins and color of diverse tomato genotypes. J. Agric. Food Chem. 2011; 59:11676–11682.
Borghesi E, Ferrante A, Gordillo B, Rodríguez-Pulido FJ, Cocetta G, Trivellini A, Mensuali-Sodi A, Malorgio F, Heredia FJ. Comparative physiology during ripening in tomato rich-anthocyanins fruits. Plant. Growth. Regul. 2016; 80:207–214. doi 10.1007/s10725-016-0158-y.
Fernández-Lara R, Gordillo B, Rodríguez-Pulido FJ, González-Miret ML, del Villar-Martínez AA, Dávila-Ortiz G, Heredia FJ. Assessment of the differences in the phenolic composition and color characteristics of new strawberry (Fragaria x ananassa Duch.) cultivars by HPLC–MS and imaging tristimulus colorimetry. Food Res. Int. 2015; 76:645–653.
He F, Mu L, Yan G-L, Liang N-N, Pan Q-H, Wang J, Reeves MJ, Duan C-Q. Biosynthesis of anthocyanins and their regulation in colored grapes. Molecules. 2010; 15(12):9057–9091.
Tanaka Y, Brugliera F, Kalc G, Senior M, Dyson B, Nakamura N, Katsumoto Y, Chandler S. Flower color modification by engineering of the flavonoid biosynthetic pathway: practical perspectives. Biosci. Biotechnol. Biochem. 2010; 74(9):1760–1769.
Silva S, Costa Em, Calhau C, Morais Rm, Pintado Me. Anthocyanin extraction from plant tissues: a review. Crit. Rev. Food Sci. Nutr. 2015; doi:10.1080/10408398.2015.1087963.
Petrov V, Slavcheva S, Stanimirov S, Pina F. Origin of the metastable statility in flavylium multistate systems. J. Phys. Chem. A. 2015; 119(12):2908–2918.
Pina F. Anthocyanins and related compounds. Detecting the change of regime between rate control by hydration or by tautomerization. Dyes Pigments. 2014; 102:308–314.
Prior RL, Wu X. Anthocyanins: structural characteristics that result in unique metabolic patterns and biological activities. Free Radical Res. 2006; 40(10):1014–1028.
Zhao C-L, Yu Y-Q, Chen Z-J, Wen G-S, Wei F-G, Zheng Q, Wang C_D, Xiao X-L. Stability-increasing effects of anthocyanin glycosyl acylation. Food Chem. 2017; 214:119–128.
Heredia FJ, Francia-Aricha EM, Rivas-Gonzalo JC, Vicario IM, Santos-Buelga C. Chromatic characterization of anthocyanins from red grapes I. pH effect. Food Chem. 1998; 63(4):491–498.
Kahkonen MP, Heinonen M. Antioxidant activity of anthocyanins and their aglycons. J. Agric. Food Chem. 2003; 51(3):628–633.
Giusti MM, Rodríguez-Saona LE, Griffin D, Wrolstad RE. Electrospray and tandem mass spectroscopy as tools for anthocyanin characterization. J. Food Agric. Chem. 1999; 47(11):4657–4664.
McCallum JL, Yang R, Young JC, Strommer JN, Tsao R. Improved high performance liquid chromatographic separation of anthocyanin compounds from grapes using a novel mixed-mode ion exchange reverse-phase column. J. Chromatogr. A. 2007; 1148(1):38–45.
Clifford MN. Anthocyanins – nature, occurrence and dietary burden. J. Sci. Food Agric. 2000; 80(7):1063–1072.
Manganaris GA, Goulas V, Vicente AR, Terry LA. Berry antioxidants: small fruits providing large benefits. J. Sci. Food Agric. 2014; 94(5):815–833.
Jimenez-Garcia S, Guevara-Gonzalez RG, Miranda-Lopez R, Feregrino-Perez A, Torres-Pacheco I, Vazauez-Cruz MA. Functional properties and quality characteristics of bioactive compounds in berries: biochemistry, biotechnology and genomics. Food Res. Int. 2013; 54(1):1195–1207.
Norberto S, Silva S, Meireles M, Faria A, Pintado M, Calhau, C. Blueberry anthocyanins in health promotion: a metabolic overview. J. Funct. Foods. 2013; 5(4):1518–1528.
Horbowicz M, Kosson R, Grzesink A, Debski H. Anthocyanin of fruits and vegetables – their occurrence, analysis and role in human nutrition. Veg. Crops Res. Bullet. 2008; 68:5–22.
Wang LS, Stoner GD. Anthocyanins and their role in cancer prevention. Cancer Lett. 2008; 269(2):281–290.
Quina FH, Moreira PF, Vautier-Giongo C, Rettori D, Rodrigues RF, Freitas AA, Silva PF, Maçanita AL. Photochemistry of anthocyanins and their biological role in plant tissues. Pure Appl. Chem. 2009; 81(9):1687–1694.
Tsao R. Chemistry and biochemistry of dietary polyphenols. Nutrients. 2010; 2:1231–1246.
Andersen OM. Recent advances in the field of anthocyanins—main focus on structures. In Recent Advances in Polyphenol Research. Daayf F, Lattanzio V. (Eds.), Blackwell Publishing: Chichester, UK, Vol. 1, 2001. pp. 167–201.
Ghosh D, Konishi T. Anthocyanin and anthocyanin-rich extracts: role in diabetes and eye function. Asia Pac. J. Clin. Nutr. 2007; 16(2):200–208.
Ali HM, Almagribi W, Al-Rashidi MN. Antiradical and reluctant activities of anthocyanidins and anthocyanins, structure-activity relationship and synthesis. Food Chem. 2016; 194:1275–1282.
Cassidy A, Bertoia M, Chiuve S, Flint A, Forman J, Rimm EB. Habitual intake of anthocyanins and flavanones and risk of cardiovascular disease in men. Am. J. Clin. Nutr. 2016; 104:587–594.
Liobikas J, Skemiene K, Trumbeckaite S, Borutaite V. Anthocyanins in cardioprotection: a path through mitochondria. Pharmacol. Res. 2016; 113:808–815.
Faria A, Fernandes I, Norberto S, Mateus N, Clhau C. Interplay between anthocyanins and gut microbiota. J. Agric. Food Chem. 2014; 62:6898–6902.
Chahar MK, Sharma N, Dobhal MP, Yoshi YC. Flavonoids: a versatile source of anticancer drugs. Pharmacogn. Rev. 2011; 5(9):1–12.
Liu Z-Q. Chemical methods to evaluate antioxidant ability. Chem. Rev. 2010; 110(10):5675–5691.
Lila MA, Burton-Freeman B, Grace M, Kalt W. Unraveling anthocyanin bioavailability for human health. Annu. Rev Food Sci. Technol. 2016; 7:375–393.
Fang J. Bioavailability of anthocyanins. Drug Metab. Rev. 2014; 46(4):508–520.
Fernandes I, Faria A, Calhau C, de Freitas V, Mateus N. Bioavailability of anthocyanins and derivatives. J. Funct. Foods. 2014; 7:54–66.
Yang M, Si K, Wo S, Ok C. Food matrix affecting anthocyanin bioavailability: review. Curr. Med. Chem. 2011; 18(2):291–300.
Wallace TC. Anthocyanins in cardiovascular disease. Adv. Nutr. 2011; 2:1–7.
Rajasekaran A, Sivagnanam G, Xavier R. Nutraceuticals as therapeutic agents: a review. Res. J. Pharm. Technol. 2008; 1(4):328–340.
Wu X, Prior R. Anthocyanins: analysis and distribution in selected medicinal plants. Chem. Lett. 2008; 12(1):9–22.
Wallace TC, Giusti MM. Anthocyanins. Adv. Nutr. 2015; 6:20–22.
Mlodzinski E. Survey of plant pigments: molecular and environmental determinants of plant colors. Acta Biol. Crac. Ser. Bot. 2009; 51(1):7–16.
Tanaka Y, Ohmiya A. Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathway. Curr. Opin. Biotechnol. 2008; 19(2):190–197.
Mortensen A. Caratenoids and other pigments as natural colorants. Pure Appl. Chem. 2006; 78(8):1477–1491.
Sun W, Liang L, Meng X, Li Y, Gao F, Liu X, Wang S, Gao X, Wang L. Biochemical and molecular characterization of a flavonoid 3-O-glycosyltransferase responsible for anthocyanins and flavonols biosynthesis in Freesia hybrida. Front. Plant Sci. 2016; 7:1–14.
Wrolstad RE, Culver CA. Alternatives to those artificial FD&C food colorants. Annu. Rev. Food Sci. Technol. 2012; 3:59–77.
Ngamwonglumlert L, Devahastin S, Chiewchan N. Natural colorants: pigment stability and extraction yield enhancement via utilization of appropriate pretreatment and extraction methods. Crit. Rev. Anal. Chem. 2015; DOI: 10.1080/10408398.2015.1109498
Mateus N, de Freitas V. Anthocyanins as food colorants. In Anthocyanins, Biosynthesis, Functions and Applications. Gould K, Davies K, Winifield C. (Eds.), Chapter 9, New York: Springer, 2009. pp. 283–304.
Hartmanova L, Ranc V, Papouskova B, Bednar P, Havlicek V, Lemr K. Fast profiling of anthocyanins in wine by desorption nano-electrospray ionization mass spectrometry. J. Chromatogr. A. 2010; 1217(25):4223–4228.
Pisano PL, Silva, MF, Olivieri AC. Anthocyanins as markers for the classification of Argentinean wines according to botanical and geographical origin. Chemometric modelling of liquid chromatography-mass spectrometry data. Food Chem. 2015; 175:174–180.
Rentzsch M, Schwarz M, Winterhalter P. Pyranoanthocyanins – an overview on structures, occurrence and pathways of formation. Trends Food Sci. Technol. 2007; 18(10):526–534.
Trouillas P, Sancho-Garcia, JC, de Freitas V, Gierschner J, Otyepka M, Dangles O. Stabilizing and modulating color by copigmentation: insights from theory and experiment. Chem. Rev. 2016; 116(9):4937–4982.
Lambert SG, Asenstorfer RE, Williamson NM, Iland PG, Graham P. Copigmentation between malvidin-3-glucoside and some wine constituents and its importance to colour expression in red wine. Food Chem. 2011; 125(1):106–115.
Bimpilas A, Panagopoulou M, Tsimogiannis D, Oreopoulou V. Anthocyanin copigmentation and color of wine: the effect of naturally obtained hydroxycinnamic acids as cofactors. Food Chem. 2016; 197(Pt A):39–46.
Heras-Roger J, Diaz-Romero C, Darias-Martin J. What gives a wine its strong red color? Main correlations affecting copigmentation. J. Agric. Food Chem. 2016; 64(34):6567–6574.
González-Manzano S, Santos-Buelga C, Dueñas M, Rivas-Gonzalo JC, Escribano-Bailón T. Colour implications of self-association processes of wine anthocyanins. Eur. Food Res. Technol. 2008; 226(3):483–490.
Davies K. Plant pigments and their manipulation. Ann. Plant Rev. Vol. 14. Boca Raton, FL: CRC Press, 2004.
Galvano F, La Fauci L, Lazzarino G, Fogliano V, Ritieni A, Ciappellano S, Battistini NC, Tavazzi B, Galvano G. Cyanidins: metabolism and biological properties. J. Nutr. Biochem. 2004; 15(1):2–11.
Wu X, Beecher GR, Holden JM, Haytowitz DB, Gebhardt SE, Prior RL. Concentration of anthocyanins in common foods in the United States and estimation of normal consumption. J. Agric. Food Chem. 2006; 54(11):4069–4075.
Drossard C, Bolzenius K, Kunz C, Kersting M. Anthocyanins in the diet of children and adolescents: intake, sources and trends. Eur. J. Nutr. 2013; 52(2):667–76.
United States Department of Agriculture. Agricultural Research Service.http://www.ars.usda.gov/
Drossard C, Fröhling B, Dietrich H, Kersting M. Anthocyanin analysis in banana fruit. A mistake. Am. J. Clin. Nutr. 2011; 93:865–866.
Jing P, Giusti MM. Contribution of berry anthocyanins to their chemopreventive properties. In Berries and Cancer Protection. Stoner GD, Seeram, NP. (Eds.), Chapter 1. New York: Springer, 2011. pp. 1–38.
Luthria DL, Natarajan SS. Influence of sample preparation of the assay of isoflavones. Planta Medica. 2009; 75(7):704–710.
Cavalcanti RN, Santos DT, Meireles MAA. Non-thermal stabilization mechanisms of anthocyanins in model and food systems: an overview. Food Res. Int. 2011; 44(2):409–509.
Baiano A, Del Nobile MA. Antioxidant compounds from vegetable matrices: biosynthesis, occurrence and extraction systems. Crit. Rev. Food Sci. Nutr. 2016; 56(12):2053–2068.
Jing P, Giusti MM. Analysis of anthocyanins in biological samples. In Anthocyanins in Health and Disease. Wallace TC, Giusti MM. (Eds.), Chapter 4. Boca Raton, FL: Taylor and Francis, 2014. pp. 115–139.
Fontana AR, Antoniolli A, Bottini R. Grape pomace as sustainable source of bioactive compounds: extraction, characterization and biotechnological applications of phenolics. J. Agric. Food Chem. 2013; 51:8987–9003.
Kirby CW, Wu T, Tsao R, McCallum JL. Isolation and structural characterization of unusual pyranoanthocyanins and related anthocyanins from Staghorn sumac ( Rhus typhinaL.) via UPLC-ESI-MS, (1)H, (13)C and 2D NMR spectroscopy. Phytochem. 2013; 94:284–293.
Li D, Wang P, Luo Y, Zhao M, Chen F. Health benefits of anthocyanins and molecular mechanisms: update from recent decade. Crit. Rev. Food Sci. Nutr. 2015; DOI: 10.1080/10408398.2015.1030064.
Jordheim M. Isolation, identification and properties of pyranoanthocyanins and anthocyanin forms. PhD Thesis. Department of Chemistry, University of Bergen, 2007.
Luthria DL. Significance of sample preparation in developing analytical methodologies for accurate estimation of bioactive compounds in functional foods. J. Sci. Food Agric. 2006; 86(14):2266–2272.
Dai J, Mumper RJ. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules. 2010; 15(10):7313–7352.
Rijke E de, Out P, Niessen WMA, Ariese F, Gooier C, Brinkman UA. Analytical separation and detection methods for flavonoids. J. Chromatogr. A. 2006; 1112(1–2):31–63.
Strack D, Heilemann J, Wray V, Dirks H. Structures and accumulation patterns of soluble and insoluble phenolics from Norway spruce needles. Phytochem. 1989; 28(8):2071–2078.
Tura D, Robards K. Sample handling strategies for the determination of biophenols in food and plants. J. Chromatogr. A. 2002; 975(1):71–93.
Rijke E de. Trace-level Determination of Flavonoids and Their Conjugates. Application to Plants of the Leguminosae Family. Amsterdam: Academisch Proefschrift, Wrije University, 2005.
Zou TB, Wang M, Gan RY, Ling WH. Optimization of ultrasound-assisted extraction of anthocyanins from mulberry, using response surface methodology. Int. J. Mol. Sci. 2011; 12(5):3006–3017.
Welch CR, Wu Q, Simon JE. Recent advances in anthocyanins analysis and characterization. Curr. Anal. Chem. 2008; 4(2):75–101.
Mazza G, Cacace JE, Kay DC. Methods of analysis for anthocyanins in plants and biological fluids. J. AOAC Int. 2004; 87(1):129–145.
Shi J, Nawaz H, Pohorli J, Mittal G, Kakuda Y, Kiang Y. Extraction of polyphenolics from plant material for functional foods-engineering and technology. Food Rev. Int. 2005; 21(1):139–166.
Nicoue EE, Savards S, Belkacemi K. Anthocyanins in wild blueberries of Quebec: extraction and identification. J. Agric. Food Chem. 2007; 55(14):5625–5635.
Liu EH, Qi LW, Cao J, Li, P. Li, CY, Peng YB. Advances of modern chromatographic and electrophoretic methods in separation and analysis of flavonoids. Molecules. 2008; 13(10):2521–2544.
Asada T, Koi Y, Arakawa R, Zhu F, Sadaoka M, Tamura H. Isolation techniques for anthocyanidin 3,5-diglucosides and their related chemicals using supramolecules technique and two solid-phase extraction cartridges. J. Chromatogr. A. 2014; 1351:21–28.
de Ferrars RM, Czank C, Saha S, Needs PW, Zhang Q, Saki Raheem K, Botting NP, Kroon PA, Kay CD. Methods for isolating, identifying and quantifying anthocyanin metabolites in clinical samples. Anal. Chem. 2014; 86:10052–10058.
Ferreiro-González M, Carrera C, Ruiz-Rodríguez A, Barbero GF, Ayuso J, Palma M, Barroso CG. A new solid phase extraction for the determination of anthocyanins in grapes. Molecules. 2014; 19:21398–21410.
Ivanova Petropulos V, Dörnyei A, Stefova M, Stafilov T, Vojnoski B, Márk L, Hermosín-Gutiérrez I, Kilár K. Application of a novel small-scale sample cleanup procedure prior to MALDI-TOF-MS for rapid pigment fingerprinting of red wines. Food Anal. Methods. 2014; 7:820–827.
Wang Y, Liu Y, Xiao C, Liu L, Hao M, Wang J, Liu X. simultaneous determination of 15 phenolic constituents of Chinese black rice wine by HPLC-MS/MS with SPE. J. Food Sci. 2014; 79:1100–1105.
Delgado de la Torre MP, Ferreiro-Vera C, Priego-Capote F, Luque de Castro MD. Anthocyanidins, proanthocyanidins and anthocyanins profiling in wine lees by solid-phase extraction−liquid chromatography coupled to electrospray ionization tand-em mass spectrometry with data-dependent methods. J. Agric. Food Chem. 2013; 61: 12539–12548.
Manns DC, Mansfield AK. A core-shell column approach to a comprehensive high-performance liquid chromatography phenolic analysis of Vitis viniferaL. and interspecific hybrid grape juices, wines and other matrices following either solid phase extraction or direct injection. J. Chromatogr. A. 2012; 1251:111–121.
Choi S-J, Choi J, Uk Lee C, Hee Yoon S, Kyung Bae S, Chin Y-W, Kim J, Dong Yoon K. Rapid separation of cyanidin-3-glucoside and cyanidin-3-rutinoside from crude mulberry extract using high-performance countercurrent chromatography and establishment of a volumetric scale-up process. J. Sep. Sci. 2015; 38:1828–1836.
Chen L, Xin X, Lan R, Yuan Q, Wang X, Li Y. Isolation of cyanidin 3-glucoside from blue honeysuckle fruits by high-speed counter-current chromatography. Food Chem. 2014; 152:386–390.
Sheng F, Wang Y, Zhao X, Tian N, Hu H, Li P. Separation and identification of anthocyanin extracted from mulberry fruit and the pigment binding properties toward human serum albumin. J. Agric. Food Chem. 2014; 62:6813−6819.
Li S, Guo L, Liu C, Fu Z, Zhang Y. Combination of supercritical fluid extraction with counter-current chromatography to isolate anthocyanidins from the petals of Chaenomeles sinensisbased on mathematical calculations. J. Sep. Sci. 2013; 36: 3517–3526.
Heinone J, Farahmandazad H, Vuorinen A, Kallio H, Yang B, Sainio T. Extraction and purification of anthocyanins from purple-fleshed potato. Food Bioprod. Process. 2016; 99:136–146.
Wen Y, Chen H, Zhou X, Deng Q, Zhao C, Gong X. A polyamide resin based method for adsorption of anthocyanins from blackberries. New J. Chem. 2016; 40:3773–3780.
Yao L, Zhang N, Wang C, Wang C. Highly selective separation and purification of anthocyanins from bilberry based on a macroporous polymeric adsorbent. J. Agric. Food Chem. 2015; 63:3543−3550.
Buran TJ, Sandhu AK, Li Z, Rock CR, Yang WW, Gu L. Adsorption/desorption characteristics and separation of anthocyanins and polyphenols from blueberries using macroporous adsorbent resins. J. Food Eng. 2014; 128:167–173.
Jampani C, Naik A, Raghavarao KSMS. Purification of anthocyanins from jamun ( Syzygium cuminiL.) employing adsorption. Sep. Purific. Technol. 2014; 125:170–178.
D’Alessandro LG, Vauchel P, Przybylski R, Chataigné G, Nikov I, Dimitrov K. Integrated process extraction-adsorption for selective recovery of antioxidant phenolics from Aronia melanocarpaberries. Sep. Purific. Technol. 2013; 120:92–101.
Ozgen S, Sarıoglu K. Synthesis and characterization of acrylonitrile-co- divinylbenzene (AN/DVB) polymeric resins for the isolation of aroma compounds and anthocyanins from strawberry. Food Bioprocess. Technol. 2013; 6:2884–2894.
Sandhu AK, Gu L. Adsorption/desorption characteristics and separation of anthocyanins from muscadine ( Vitis rotundifolia) juice pomace by use of macroporous adsorbent resins. J. Agric. Food Chem. 2013; 61:1441–1448.
Sarıoglu K, Erdem T, Ozgen S. Synthesis and characterization of lipophilic polymeric adsorbent resins for recovery of anthocyanins from black carrot. Food Anal. Methods. 2013; 6:1621–1628.
Da Fonseca Machado AP, Pasquel-Reátegui JL, Fernández Barbero G, Martínez J. Pressurized liquid extraction of bioactive compounds from blackberry ( Rubus fruticosusL.) residues: a comparison with conventional methods. Food Res. Int. 2015; 77:675–683.
Otero-Pareja MJ, Casas L, Fernández-Ponce MT, Mantell C, Martínez de la Ossa EJ. Green extraction of antioxidants from different varieties of red grape pomace. Molecules. 2015; 20:9686–9702.
Casas Cardoso L, Mantell Serrano C, Torrez Quintero E, Pereyra López C, Medrano Antezana R, Martínez de la Ossa EJ. High pressure extraction of antioxidants from Solanum stenotomun peel. Molecules. 2013; 18:3137–3151.
Santos DT, Veggi PC, Meireles MA. Optimization and economic evaluation of pressurized liquid extraction of phenolic compounds from jabuticaba skins. J. Food Eng. 2012; 108:444–452.
Truong VD, Hu Z, Thompson RL, Yencho GC, Pecota KV. Pressurized liquid extraction and quantification of anthocyanins in purple-fleshed sweet potato genotypes. J. Food Compos. Anal. 2012; 26:96–103.
Wen Y, Chen H, Zhou X, Deng Q, Zhao Y, Zhao C, Gong X. Optimization of the microwave-assisted extraction and antioxidant activities of anthocyanins from blackberry using a response surface methodology. RSC Adv. 2015; 5:19686–19695.
Routray W, Orsat V. MAE of phenolic compounds from blueberry leaves and comparison with other extraction methods. Ind. Crop. Prod. 2014; 58:36–45.
Pap N, Beszédes S, Pongrácz E, Myllykoski L, Gábor M, Gyimes E, Hodúr C, Keiski RL. Microwave-assisted extraction of anthocyanins from black currant marc. Food Bioprocess Tech. 2013; 6:2666–2674.
Zou T, Wang D, Guo H, Zhu Y, Luo X, Liu F, Ling W. Optimization of microwave-assisted extraction of anthocyanins from mulberry and identification of anthocyanins in extract using HPLC-ESI-MS. J. Food Sci. 2012; 71:46–50.
Picariello G, Ferranti P, Garro G, Manganiello G, Chianese L, Coppola R, Addeo F. Profiling of anthocyanins for the taxonomic assessment of ancient purebred V. Viníferared grape varieties. Food Chem. 2014; 146:15–22.
Gardana C, Ciappellano S, Marinoni L, Fachechi C, Simonetti P. Bilberry adulteration: identification and chemical profiling of anthocaynins by different analytical methods. J. Agric. Food Chem. 2014; 45:10998–11004.
Barnes JS, Nguyen HP, Shen S, Schug KA. General method for the extraction of blueberryanthocyanins and identification using high performance liquid chromatography- electrospray ionization-ion trap-time of flight-mass spectrometry. J. Chromatogr. A 2009; 16(23):4728–4735.
Jin H, Liu Y, Yang F, Wang J, Fu D, Zhang X, Peng X, Liang X. Characterization of anthocyanins in wild Lycium tuthenium Murray by HPLC-DAD/QTOF-MS/MS. Anal. Meth. 2015; 7:4947–4956.
Scotter MJ. Emerging and persistent issues with artificial food colour: natural colour additives as alternatives to synthetic colour in food and drink. Qual. Assurance Saf. Crops Foods 2011; 3(1):28–39.
Scotter MJ. Methods for the determination of European union-permitted added natural colours in foods: a review. Food Addit. Colorants Part A. 2011; 28:527–596.
Scotter, M. Review and evaluation of available methods of extraction and analysis for approved natural colours in food and drink; Report Number FD10/02. London: Food Standard Agency, March 2010.
Ferrari E, Foca G, Vignali M, Tassi L, Ulrich A. Adulteration of the anthocyanin content of red wines: perspectives for authentication by Fourier transform-near infrared and 1HNMR spectroscopies. Anal. Chim. Acta. 2011; 701(2):139–151.
Flamini R. Recent applications of mass spectrometry in the study of grape and wine polyphenols. ISRN Spectroscopy 2013; 2013:1–45.
Li SY, He F, Zhu BQ, Xing RR, Reeves MJ, Duan CQ. A systematic analysis strategy for the accurate detection of anthocyanin pigments in red wines. Rapid Commm. Mass Spectrom. 2016; 30(13):1619–1626.
Song W, Wang HJ, Bucheli P, Zhang PF, Wei DZ, Lu YH. Phytochemical profiles of different mulberry ( Morussp.) species from China. J. Agric. Food Chem. 2009; 57(19):9133–9140.
Paredes-López O, Cervantes-Ceja ML, Vigna-Perez M, Hernandez-Perez T. Berries: improving human health and healthy aging and promoting quality life. A review. Plant Foods Hum. Nutr. 2010; 65(3):299–308.
Benvenuti S, Bortolotti E, Maggini R. Antioxidant power, anthocyanin content and organoleptic performance of edible flowers. Sci. Hort. 2016; 199(16):170–177.
Smeriglio A, Bareca D, Belloccoco E, Trombetta D. Chemistry, pharmacology and health benefits of anthocyanins. Phytother. Res. 2016; 30(8):1256–1286.
Li X, Ma H, Huang H, Li D, Yao S. Natural anthocyanins from phytoresources and their chemical researches. Nat. Prod. Res. 2013; 27(4–5):456–469.
Iwashina T. Contribution to flower colors of flavonoids including anthocyanins: a review. Nat Prod. Commun. 2015; 10(3):529–544.
Kuskoski EM, Vega JM, Rios JJ, Fett R, Troncoso AM, Asuero AG. Characterization of anthocyanins from the fruits of baguaçu ( Eugenia umbellifloraBerg). J. Agric. Food Chem. 2003; 51(18):5450–5454.
Kuskoski EM, Asuero AG, Morales MT, Fett R. Wild tropical fruits and frozen fruit pulps: antioxidant activity, polyphenols and anthocyanins. (in Portuguese) Ciência Rural. 2006; 36(4):1283–1287.
Kuskoski EM, Asuero AG, García-Parrilla MC, Troncoso AM, Fett R. Actividad antioxidante de pigmentos antociánicos. Cienc. Tecnol. Campinas. 2005; 24(4):691–693.
Kuskoski EM, Asuero AG, Troncoso AM, Mancini-Filho J, Fett , R. Application of different chemical methods to determine antioxidant activity in fruit pulp. (in Spanish) Food Sci. Technol. 2005; 25(4):726–732.
Veitch, NC, Grayer RJ. Flavonoids and their glycosides, including anthocyanins. Nat. Prod. Rep. 2011; 28(10):1626–1695.
Yousuf B, Gul K, Wani AA, Singh P. Health benefits of anthocyanins and their encapsulation for potential use in food systems; a review. Crit. Rev. Food Sci. Nutr. 2016; 56(13):2223–2230.
Robert P, Fredes C. The encapsulation of anthocyanins from berry-type fruits. Trends in foods. Molecules. 2015; 20(4):5875–5888.
Rodriguez-Saona L, Wrolstad RE. Extraction, isolation and purification of anthocyanins. Curr. Protoc. Food Anal. Chem. 2001;F1.1–F1.11.
Bucar F, Wube A, Schmid M. Natural product, isolation. How to get from biological material to pure compounds. Nat. Prod. Rep. 2013; 30:525–545.